Although ₂-adrenoceptors represent 15-25 % of -adrenoceptors in the guinea-pig heart, their functionality is controversial. We assessed the inotropic effects of ₂-adrenoceptor partial agonists in right papillary muscles. Salbutamol induced a small but significant concentration-dependent negative inotropic effect (NIE, -5 % at 60 nM) followed by a moderate positive inotropic effect (+36 % at 6 µM) due to activation of 1-adrenoceptors. In the presence of 4 µM atenolol, the concentration-dependent NIE (-12 % at 6 µM) was biphasic, best described by a double logistic equation with respective EC₅₀ of 3 and ~420 nM and was insensitive to SR59230A. In muscles from pertussis toxin-treated guinea-pigs, the salbutamol-induced positive inotropic effect was sensitive to low concentrations of ICI 118,551 in an unusual manner. Experiments in reserpinized animals revealed the importance of the phosphorylation-dephosphorylation processes. PKA inhibition reduced and suppressed the effects obtained at low and high concentrations, respectively, indicating that its activation was a pre-requisite to the NIE. The effect occurring at nanomolar concentrations depended upon PKA/PI₃K/cPLA₂ activations leading to NO release via the arachidonic acid/COX pathway. NO release via PKA-dependent phosphorylation of the receptor was responsible for the inotropic effect observed at sub-micromolar concentrations which is negatively controlled by cPLA₂. The possibility that these effects are due to an equilibrium between different affinity states of the receptor (Gs/Gi coupled and Gi-independent with different signaling pathways) that can be displaced by ICI 118,551 is discussed. We conclude that ₂-adrenoceptors are functional in guinea-pig heart and can modulate the inotropic state.